We have been exploring the Hypothesis (number 1) that the ancient and divergent classes of plant genes have been preserved throughout vascular plant evolution because they have unique patterns of regulation and/or encode proteins with distinct functions. We made use of the compact genome, rapid life cycle, small size, ease of transformation and regeneration, and simple genetics of Arabidopsis to dissect different parts of this hypothesis. Based on the analysis of actin promoter/reporter fusions in hundreds of transgenic plants, RNA levels, and protein levels assayed with isovariant-specific monoclonal antibodies we determined that the three vegetative actins and five reproductive actins each have strong and distinct spatial and temporal expression patterns. These range from constitutive expression of ACT2 in most vegetative tissues, to the phytohormonal induction of ACT7, to the expression of ACT1 in ovules and pollen. The first sequence-based method of isolating plant insertion mutants was developed and used to isolate mutants in four of the eight- expressed actin genes. These mutants have exciting and subtle cell and developmental phenotypes that are revealed when plants are grown under specialized conditions. Multigenerational studies on mutants in three actin genes demonstrated that each gene is under strong selective constraint and is required for the survival of Arabidopsis. The extreme sequence diversity of the plant actin isovariants and their complex expression patterns lead to a second hypothesis. Hypothesis number 2 states that the coexpression of multiple actin isovariants in the same cell results in isovariant dynamics that allow for the temporal and biochemical expansion and buffering of responses of a biological system. A wide variety of molecular, cellular, genetic, and biochemical approaches are needed to address the following Specific Aims: 1) to isolate mutants in each actin and profilin gene; 2) to characterize the phenotypes of these mutants; 3) to determine the spatial or temporal expression patterns of the actin isovariants; 4) to determine the subcellular distribution of coexpressed actins; 5) to dissect the physical/molecular parameters that distinguish the actin isovariants using the functional actin proteins being produced in yeast.